Version 05.29.2013
Protein-Protein Conjugation Kit
Technical Manual
Catalog # S-9010-1
Note: This protocol and any documents linked below can be downloaded from the appropriate category in the Solulink Library at
http://www.solulink.com/library.
1
Disclaimer
The products offered here are for research use only. Any commercial application will require a license from
Solulink. The Solulink Conjugation System is patented and has multiple patents pending. Please contact
Solulink for information regarding licensing information. Solulink products and methods may be covered by
one or more of the following United States patents Nos. 6,686,461, 6,800,728, 7,102,024, 7,173,125,
7,462,689 and other pending patent applications. Information in this manual is subject to change without
notice and does not constitute a commitment on the part of Solulink, Inc. It is supplied on an “as is” basis
without any warranty of any kind, either explicit or implied. Information may be changed or updated in this
manual at any time. This document may not be copied, transferred, reproduced, disclosed, or duplicated, in
whole or in part, without the prior written consent of Solulink, Inc. This documentation is proprietary
information and protected by the copyright laws of the United States and international treaties. The
manufacturer of this documentation is Solulink, Inc.
Safety Information
WARNING – CHEMICAL HAZARD. Some chemicals used can be potentially hazardous, and can cause injury
or illness.
•
Read and understand the Material Safety Data Sheets (MSDS) available at Solulink.com before you store,
handle, or work with any chemicals or hazardous materials.
•
Minimize contact with and inhalation of chemicals. Wear appropriate personal protective equipment
when handling chemicals (e.g. safety glasses, gloves, or clothing). For additional safety guidelines
consult the MSDS.
•
Check regularly for chemical leaks or spills. If a leak or spill occurs, follow the manufacturer’s clean-up
procedures as recommended in the MSDS.
•
Comply with all local, state/provincial, or national laws and regulations related to chemical storage,
handling and disposal.
2
Table of Contents
Introduction to the Solulink Bioconjugation Technology................................................................................................. 4
The Keys to Successful Conjugation ................................................................................................................................. 5
Kit Components................................................................................................................................................................ 6
Equipment/Regents Required but Not Provided ............................................................................................................. 6
Protein-Protein Conjugation Protocol.............................................................................................................................. 6
2.0 Protein Modification ........................................................................................................................................... 9
2.1 S-HyNic-Protein Modification Protocol (Calculator Worksheet 1) ...................................................................... 9
2.2 Determining the HyNic Molar Substitution Ratio (MSR)................................................................................... .10
2.3 S-4FB Protein Modification Protocol (Calculator Worksheet 2) ........................................................................ .11
2.4 Determining the 4FB Molar Substitution Ratio (MSR)…………………………….……..………………….…….…………………… .12
3.0 Protein-Protein Conjugation (Calculator Worksheet 3) ..................................................................................... .13
4.0 Purification…………………………………………………………………………………………………………………………………..……………. ...14
5.0 Troubleshooting……………………………………………………………………………………………………………………………………..…… ..15
6.0 Appendix………………………………………………………………………………………………………………………………………………….….. .16
3
Introduction to the Solulink Bioconjugation Technology
The Reaction:
SoluLinK’s core technology is based on the formation of a stable bis-arylhydrazone formed from an aromatic
hydrazine and an aromatic aldehyde. S-HyNic (succinimidyl 6-hydrazinonicotinate acetone hydrazone, SANH)
is used to incorporate aromatic hydrazine moieties on biomolecules. S-HyNic is an amino-reactive reagent
that directly converts amino groups on biomolecules and surfaces to HyNic groups. S-4FB (succinimidyl 4formylbenzoate, SFB) is used to convert amino groups to aromatic aldehydes (4-formylbenzamide (4FB)
groups). Addition of a HyNic-modified biomolecule to a 4FB-modified biomolecule or surface directly leads
to the formation of the conjugate (Figure 1). The bis-arylhydrazone bond is stable to 92OC and pH 2.0-10.0.
Due to the lability of the immunoreactivity of antibodies at low pH, i.e. < 5.0, the recommended pH for
antibody conjugation is 6.0. Unlike thiol-based conjugation protocols where reducing reagents are required
that can compromise the activity of proteins by cleaving disulfide bonds, the HyNic-4FB conjugation couple
leaves disulfide bonds intact. No oxidants, reducing agents, or metals are required in the preparation of
conjugate.
Figure 1: Linking chemistry behind HydraLink
TM
technology.
Fastest, most efficient:
Further enhancing the many advantages of the HyNic/4FB conjugation couple is the discovery by Dirksen et
al. that showed aniline catalyzes the formation of this Schiff’s base. This is especially effective for large
biomolecule conjugations. In the case of antibody-protein conjugations the addition of 10 mM aniline to the
reaction mixture converts >95% of the antibody to conjugate in 2 hours using 1-2 mole equivalents of
second protein.
Traceable modification:
Reproducibility of any reaction is dependent on accurate characterization of all components. As both HyNic
and 4FB are aromatic, their incorporation can be readily quantified using colorimetric assays.
Traceable conjugation:
The HyNic-4FB conjugate bond is chromophoric. It absorbs at 354 nm and has a molar extinction coefficient
of 29,000.
Furthermore, compared to previous methods, the HyNic/4FB technology offers the following practical
advantages:
4
1) The reaction goes to completion: In all previous bi-functional linker based conjugations, the reaction
never went to completion, i.e. there was always unconjugated limiting protein in the final reaction. The
HyNic-4FB conjugation couple catalyzed by aniline yields more than 90% conjugate.
2) The reaction is efficient: The reaction is very stoichiometrically efficient as input of only 1-2 mole
second protein/mole first protein is required for complete conversion to conjugate.
3) The conjugate bond is extremely stable: The bis-arylhydrazone conjugate bond is stable to 92OC and pH
2.0-10.0.
4) The reaction conditions are extremely mild and do not cause antibody denaturation: Unlike thiolbased conjugation protocols where reducing reagents are required that can compromise the activity of
proteins by cleaving disulfide bonds, the HyNic-4FB conjugation couple leaves disulfide bonds intact. No
metals, oxidizing, or reducing reagents are required.
5) The conjugation is traceable spectrophotometrically. The HyNic-4FB conjugate bond is chromophoric; it
absorbs at 354 nm and has a molar extinction coefficient of 29,000.
6) The modifications of both the HyNic moiety on the protein and the 4FB moiety on the protein is
quantifiable using a colorimetric assay. The reproducibility of any reaction is dependent on accurate
characterization of all components. The Molar Substitution Ratio (MSR), i.e. the number of HyNic
moieties/protein, of HyNic groups can be quantified colorimetrically as reaction with 2sulfobenzaldehyde yields a chromophoric product that absorbs at 348 nm with a molar extinction
coefficient of 28,500. The MSR of 4FB groups can be determined colorimetrically by its reaction with 2hydrazinopyridine forming a hydrazone that absorbs at 354 nm with a molar extinction coefficient of
24,500. This kit contains all the reagents necessary to determine both MSRs. Procedures to guide users
through this process are given in the protocol below.
The Keys to Successful Conjugation
The following are three crucial requirements that must be fulfilled for a reproducibly successful preparation
of a protein/protein conjugate using SoluLink’s bioconjugation technology:
1. Desalting: Prior to modification, the starting protein must be thoroughly desalted, removing all amine
contaminants, and exchanged into 1X Modification Buffer.
2. Protein concentration: The recommended concentration of protein must be adhered to in all steps.
3. Molar substitution ratio: The molar ratio of HyNic on the protein and 4FB on the protein must be
determined and within the desired range before continuing to the next step.
5
Kit Components
Component
S-HyNic
S-4FB
10X Modification Buffer
10X Conjugation Buffer
10X TurboLink Catalyst Buffer
7kDa 0.5 mL Zeba Columns
Anhydrous DMF
0.5 mM 2-Hydrazinopyridine reagent
0.5 mM 2-Sulfobenzaldehyde reagent
2.0 mL Collection Tubes
7kDa 2.0 mL Zeba Columns
10X PBS
Component #
S-9010-1-01
S-9010-1-02
S-9010-1-03
S-9010-1-04
S-9010-1-05
S-9010-1-06
S-9010-1-07
S-9010-1-08
S-9010-1-09
S-9010-1-11
S-9010-1-13
S-9010-1-14
Size
2 x 0.5 mg
2 x 0.5 mg
1.5 mL
1.5 mL
1.5 mL
10
2 x 1.5 mL
1 x 0.5 mL
1 x 0.5 ml
10
2
1.5 mL
Storage
Desiccated
Desiccated
4oC
4oC
4oC
4oC
Desiccated
4oC
4oC
Room temp
4oC
4oC
NOTES:
1) For convenience all kit components can be stored at 4OC.
If precipitate is present in buffers upon storage at 4OC, re-dissolve by warming at 37OC before using.
2) 10X Modification Buffer: 1.0 M phosphate, 1.5 M NaCl, pH 8.0
3) 10X Conjugation Buffer: 1.0 M phosphate, 1.5 M NaCl, pH 6.0
4) 10X TurboLink Catalyst Buffer: 100 mM aniline, 100 mM phosphate, 150 mM NaCl, pH 6.0
Equipment/Regents Required But Not Provided
1) NanoDropTMor UV-Vis Spectrophotometer
2) Protein concentration assay reagents such as BCA or Bradford assay
3) Microcentrifuge
Protein-Protein Conjugation Protocol
Over the years, Solulink’s scientists have accumulated extensive protein-protein conjugation experience.
Based on this experience, Solulink has developed and optimized conjugation protocols that work well.
Experience has taught us that certain strict limitations need to be placed on initial buffer composition,
starting mass (mg) and concentrations (mg/mL). As a consequence, before starting a conjugation project we
recommend the use of the flow chart outlined in Figure 2. To use the chart, simply start at the box labeled
‘your protein’ and proceed to answer the questions in the flow chart. The chart eventually guides the user to
the first step in the HyNic or 4FB conjugation protocol.
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Conjugation kit parameters:
1.
2.
3.
4.
Protein molecular weight range 25,000 – 950,000 Daltons
Protein concentration range 1.0 – 5.0 mg/mL; mass of protein to be modified range 50 – 650 g
Protein sample volume range 50 – 130 L
Linker mole equivalents range 3 – 20 fold
Your protein
No
Determine protein
concentration (BCA
Bradford or A280nm)
Is the protein
concentration known?
Liquid
Solid
Is the protein in liquid or solid form?
Is the available mass
between 0.05 – 0.65 mg?
Yes
Yes
Yes
Is the concentration
between 1 – 5 mg/mL?
No
Concentrate the sample to 50130 µL and 1 – 5 mg/mL
or
Dilute to 50 – 130 µL if sample
concentration is more than 5 mg/mL.
Yes
Add 1x modification buffer to
bring the protein concentration
to 1 – 5 mg/mL and volume
between 50 – 130 µL.
Yes
Yes
Is the volume
between 50 – 130µL?
No
Desalt with 0.5 mL Zeba column.
Yes
Figure 2. Flow-chart used for guiding a user to the start of the conjugation process.
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Protein Desalting
Proteins must be desalted prior to modification. Proteins must be completely desalted into 1X Modification
Buffer (dilute from 10X Modification Buffer) before they are modified with S-HyNic or S-4FB. This step ensures
that the protein is in the correct buffer for modification, and that all traces of interfering amines such as Tris,
azide, or glycine are removed.
SoluLink recommends the use of ZebaTM desalting columns (provided) to desalt proteins as required by our
conjugation protocol. These rapid spin columns are recommended because they do not significantly dilute the
protein during desalting and recover 85-90% of protein.
0.5mL
2.0mL
TM
Figure 3. Zeba desalting spin columns (0.5 and 2 mL) used to desalt
starting protein and HyNic or 4FB-modified protein.
This kit includes 0.5 mL ZebaTM desalting columns (Figure 3) that have a maximum capacity of 130 µL (and a
minimum capacity of 50 µL). Therefore up to 0.65 mg of a 5 mg/mL solution of protein can be desalted. As this
kit has been designed for two conjugations, included are eight columns, one to initially desalt each protein into
1X Modification Buffer and one to desalt and exchange the modified protein into 1X Conjugation Buffer. This kit
also includes two 0.5 mL or 2 mL ZebaTM desalting columns that have a capacity of 50 – 150 µL or 150-700 µL,
respectively, to desalt the final two protein conjugates into PBS storage buffer depending on the final reaction
volume after conjugation.
ZebaTM Column Desalting Protocol
0.5mL Zeba
1.
2.
3.
4.
5.
6.
7.
8.
TM
Spin Column Preparation (sample volumes 50-130 µL)
Remove spin column’s bottom closure and loosen the top cap (do not remove cap).
Place spin column in a 2.0 mL microcentrifuge collection tube.
Centrifuge at 1,500 x g for 1 minute; discard the flow through from the collection tube.
Place a mark on the side of the column where the compacted resin is slanted upward. Place the column
in the centrifuge with the mark facing outward and away from the center of the rotor in all subsequent
centrifugation steps.
Add 300 µL of required buffer to the top of the resin bed.
Centrifuge at 1,500 x g for 1 minute; discard the flow through from the collection tube.
Repeat steps 5 and 6 two additional times, discarding the flow through from the collection tube.
Column is now ready for sample loading.
8
Protein Sample Loading
1. Place the equilibrated spin column in a new 2.0 mL microcentrifuge tube. Remove cap and slowly apply 50130 µL sample onto the center of the compact resin bed. Avoid contact with the tube walls — the sample must
channel down through the resin itself.
2. Centrifuge at 1,500 x g for 2 minutes to collect desalted sample.
3. Protein sample is desalted and ready for the next step.
2.0 Protein Modification
Note- Modification of HyNic/protein-1 and 4FB/protein-2 can be done at the same time. This way the
whole procedure can be finished in one day. We recommend that the HyNic-modified protein be used
within 5 hours to avoid forming a dimer with itself. 4FB-modified protein is stable at 4oC for weeks.
Recommended Guidelines for Modifying Proteins with S-HyNic
The modification process is a critical element of any conjugation project. For this reason, we have included a more
detailed discussion of this important step. The number of functional groups incorporated per protein molecule is
commonly referred to as the molar substitution ratio (MSR). The final MSR obtained after a modification reaction
with S-HyNic is a function of several variables that include protein concentration, number of available amino
groups on the protein (often related to M.W.), excess linker equivalents added (e.g. 5X, 10X or 20X) and reaction
pH. Table 1 presents the results of a study to determine the level of HyNic incorporation on an antibody after
adding 5X, 10X and 20X mole equivalents of S-HyNic at 1.0, 2.5 and 5.0 mg/mL antibody concentration.
Protein concentration
1.0 mg/mL
2.5 mg/mL
5.0 mg/mL
5X HyNic
1.0
3.2
4.9
10X HyNic
1.4
5.3
6.6
20X HyNic
3.0
7.9
7.8
Table 1: MSR of Modifying Bovine IgG with S-HyNic
Note - It is recommend that the MSR of HyNic-proteins is 4 - 8 for proteins greater than
100,000 Daltons, and 2-4 for proteins equal to or less than 100,000 Daltons.
In general, as the protein concentration and number of linker equivalents are increased, the molar substitution
ratio increases. Caution is recommended since over-modification can dramatically change the isoelectric point of
the protein and result in precipitation of the protein or loss of biological activity. This is especially critical with
proteins <50 kD molecular weight.
9
2.1 S-HyNic-Protein Modification Protocol (Calculator Worksheet 1)
1. Desalt protein to be modified with S-HyNic into 1X Modification Buffer (dilute 10X stock buffer 1/10 with
water) following the Zeba desalting procedure on page 8. Confirm protein concentration by read at A 280 after
initial protein desalting. Between 50 - 130 µL of protein solution at a concentration between 1 - 5 mg/mL may
be used.
2. On the S-HyNic Modification Calculator input the name, molecular weight, protein concentration, volume of
protein-1 to be modified, and the mole equivalents of HyNic used to modify protein-1 in the green fields. Add
the required volume of anhydrous DMF to a 0.5 mg vial of pre-weighed S-HyNic reagent. Pipette the solution
up and down to dissolve the pellet. The volume of DMF will be output in the pink field. If the volume of DMF
output is more than 700 L, two or more times dilution of the HyNic reagent may be required.
For example if the volume of DMF output is 5857 L, then three times dilutions are required:
(1) Make a 58.57X stock solution by dissolving S-HyNic pellet in 100 L of DMF (5857L/100L = 58.57).
(2) Next, make a 5.857X S-HyNic solution by adding 10 L of 58.57X S-HyNic solution into 90 L of DMF
and mix well (1:10 dilution).
(3) Third, make 1X S-HyNic solution by adding 10 L of 5.857X S-HyNic solution into 48.57 L of DMF and
mix well (1:5.857 dilution).
3. If protein molecular weight is greater than 50,000 Daltons, add 2.0 L of S-HyNic reagent into the desalted
protein. If protein molecular weight is equal to or less than 50,000 Daltons add 3.0 L of S-HyNic reagent into
the desalted protein as calculated using the Protein-Protein Conjugation Calculator on calculator worksheet 1.
Immediately mix well by vortexing.
4. Incubate the reaction at room temperature for at least 2.5 hours.
5. Proceed to desalt the HyNic-modified protein using a 0.5 mL ZebaTM column equilibrated with 1X Conjugation
Buffer (dilute 10X stock buffer 1/10 with water) using the Zeba desalting protocol on page 8.
2.2 Determining the HyNic Molar Substitution Ratio (MSR)
After desalting with a ZebaTM spin column to remove excess HyNic from the modification reaction, the protein
concentration is determined by using either BCA or Bradford assay (the protein concentration must not be
determined by reading A280 on a spectrophotometer at this step because of the contribution at A280 of HyNic
itself). After the HyNic-protein concentration is determined proceed to MSR assay.
Assay Protocol
1. Transfer 2 μL of HyNic-modified (desalted) protein solution (1-5 mg/mL in 1X Conjugation Buffer) to a new
2.0 mL microfuge tube containing 18 μL of SBA working solution. Prepare another reaction tube (blank)
containing 2 μL of 1X Conjugation Buffer and 18 μL of 2-SBA working solution.
o
2. Vortex to mix well, then incubate both reaction tubes at 37 C for 1 hour.
o
3. Remove the reaction tubes from 37 C and briefly centrifuge at 10,000 x g to collect any condensate. Vortex
10
both samples thoroughly before reading the A348 by one of the following methods:
Method A: NanoDropTM Method
1. Launch the NanoDropTM software and select the UV-VIS menu option. Initialize the instrument with 2 μL
water (NanoDropTM ND-1000 only).
2. Blank the NanoDropTM with 2 μL blank (Conjugation Buffer + SBA) solution and clean the pedestal.
3. Set the λ1 wavelength to 348nm. Place 2 μL of the HyNic-protein MSR reaction on the pedestal and click the
“Measure” icon. The 1.0 mm A354 absorbance will appear. Multiply this number by 10 (converts from 1 mm
to 1 cm) and then enter this value into the HyNic MSR Calculator Protein-Protein Conjugation Calculator on
calculator worksheet 1 to obtain MSR.
Method B: Cuvette Spectrophotometer (100 μL, 1-cm micro-cuvette)
1. Prepare a 1:10 dilution of blank (Conjugation Buffer + SBA) solution by adding 180 μL of deionized water into
the blank sample tube and mix well.
2. Prepare a 1:10 dilution of HyNic-protein MSR reaction solution by adding 180 μL of deionized water into the
HyNic-protein MSR reaction tube and mix well.
3. In a 1 cm, 100 μL quartz micro-cuvette, blank the spectrophotometer at 348nm with 100 μL of the blank
solution prepared step 1 above.
4. Remove the blank solution and add 100 μL of the HyNic-protein MSR sample solution from step 2 above to
the cuvette.
5. Record the 348nm absorbance value of the HyNic-protein MSR sample. Multiply this number by 10 (to
account for the 10-fold dilution) and then enter this value into the HyNic MSR Calculator Protein-Protein
Conjugation Calculator on calculator worksheet 1 to obtain MSR.
2.3 S-4FB Protein Modification Protocol (Calculator Worksheet 2)
Recommended Guidelines for Modifying Proteins with S-4FB
Protein concentration
1.0 mg/mL
2.5 mg/mL
5.0 mg/mL
5X S-4FB
3.7
4.8
5.6
10X S-4FB
4.3
7.3
8.6
20X S-4FB
9.8
14.0
14.3
Table 2: MSR of Modifying Bovine IgG with S-4FB
Note - It is recommend that the MSR of 4FB-proteins is 4 - 8 for proteins greater than
100,000 Daltons, and 2-4 for proteins equal to or less than 100,000 Daltons.
1. Desalt protein to be modified with S-4FB into 1X Modification Buffer (dilute 10X stock buffer 1/10 with
water) following the Zeba desalting procedure on page 8. Confirm protein concentration by read at A 280 after
initial protein desalting. Between 50 - 130 µL of protein solution at a concentration between 1 - 5 mg/mL
11
may be used.
2. On the S-4FB Modification Calculator input the name, molecular weight, protein concentration, volume of
protein-2 to be modified, and the mole equivalents of S-4FB used to modify protein-2 in the green fields.
Add the required volume of anhydrous DMF to a 0.5 mg vial of pre-weighed S-4FB reagent. Pipette the
solution up and down to dissolve the pellet. The volume of DMF will be output in the pink field. If the
volume of DMF output is more than 700 L, two or more times dilution of the S-4FB reagent may be
required.
For example if the volume of DMF output is 5857 L, then three times dilutions are required:
(1) Make a 58.57X stock solution by dissolving S-4FB pellet in 100 L of DMF (5857L/100L = 58.57).
(2) Next, make a 5.857X S-4FB solution by adding 10 L of 58.57X S-4FB solution into 90 L of DMF
and mix well (1:10 dilution).
(3) Third, make 1X S-4FB solution by adding 10  of 5.857X S-4FB solution into 48.57 L of DMF and
mix well (1:5.857 dilution).
3. If the protein molecular weight is greater than 50,000 Daltons, add 2.0 L of S-4FB reagent into the desalted
protein. If protein molecular weight is equal to or less than 50,000 Daltons add 3.0 L of S-4FB reagent into
the desalted protein. Immediately mix well by vortexing.
4. Incubate the reaction at room temperature for at least 2.5 hours.
5. Proceed to desalt the 4FB-modified protein using a 0.5 mL ZebaTM column equilibrated with 1X Conjugation
Buffer (dilute 10X stock buffer 1/10 with water) using the Zeba desalting protocol on page 8.
2.4 Determining the 4FB Molar Substitution Ratio (MSR)
After desalting with a ZebaTM spin column to remove excess 4FB from the modification reaction, the protein
concentration is determined by using either BCA or Bradford assay (the protein concentration must not be
determined by reading A280 on a spectrophotometer at this step because of the contribution at A280 of 4FB
itself). After the 4FB-protein concentration is determined proceed to MSR assay.
Assay Protocol
1. Transfer 2 μL of 4FB-modified (desalted) protein solution (1-5 mg/mL in 1X Conjugation Buffer) to a new 2.0
mL microfuge tube containing 18 μL of 2-HP working solution. Prepare another reaction tube (blank)
containing 2 μL of 1X Conjugation Buffer and 18 μL of 2-HP working solution.
o
2. Vortex to mix well, then incubate both reaction tubes at 37 C for 1 hour.
o
3. Remove the reaction tubes from 37 C and briefly centrifuge at 10,000 x g to collect any condensate. Vortex
both samples thoroughly before reading the A354 by one of the following methods:
Method A: NanoDropTM Method
1. Launch the NanoDropTM software and select the UV-VIS menu option. Initialize the instrument with 2 μL
12
water (NanoDropTM ND-1000 only).
2. Blank the NanoDropTM with 2 μL blank (Conjugation Buffer + 2-HP) solution and clean the pedestal.
3. Set the λ1 wavelength to 354nm. Place 2 μL of the 4FB-protein MSR reaction on the pedestal and click the
“Measure” icon. The 1.0 mm A354 absorbance will appear. Multiply this number by 10 (converts from 1 mm
to 1 cm) and then enter this value into the 4FB MSR Calculator Protein-Protein Conjugation Calculator on
worksheet 2 to obtain MSR.
Method B: Cuvette Spectrophotometer Protocol (100 μL, 1-cm micro-cuvette)
1. Prepare a 1:10 dilution of blank (Conjugation Buffer + 2-HP) solution by adding 180 μL of deionized water
into the blank sample tube and mix well.
2. Prepare a 1:10 dilution of 4FB-protein MSR reaction solution by adding 180 μL of deionized water into the
4FB-protein MSR reaction tube and mix well.
3. In a 1 cm, 100 μL quartz micro-cuvette, blank the spectrophotometer at 354nm with 100 μL of the blank
solution prepared step 1 above.
4. Remove the blank solution and add 100 μL of the 4FB-protein MSR sample solution from step 2 above to the
cuvette.
5. Record the A354nm absorbance value of the 4FB-protein MSR sample. Multiply this number by 10 (to
account for the 10-fold dilution) and then enter this value into the 4FB MSR Calculator Protein-Protein
Conjugation Calculator on calculator worksheet 2 to obtain MSR.
3.0 Protein-Protein Conjugation (Calculator Worksheet 3)
Conjugate formation is initiated by mixing the desired equivalents of each modified protein together with
TurboLink Catalyst Buffer. Often protein-2 is added in molar excess (1 – 2 fold) over protein-1 in order to more
efficiently drive the conjugation reaction to completion.
1. Using the Protein-Protein Conjugation Calculator. On the HyNic-protein-1/4FB-protein-2 conjugation
calculator, input both protein names, molecular weights, protein concentrations, the mass of protein-1 to be
conjugated in mg, and the mole equivalents of 4FB-protein-2 to conjugate with protein-1 in the green fields.
The calculator will determine the volumes of HyNic-modified protein-1 to combine with 4FB-modified
protein-2. Mix the two indicated volumes of proteins, and add 1/10 volume of 10X TurboLink Catalyst
Buffer. The calculator will output the required volume of 10X TurboLink Catalyst Buffer at blue field.
2. Incubate the reaction at room temperature for 2-3 hours or overnight at 4oC.
3. The reaction is now ready for purification by column chromatography, if required (see section 4 below), or
for desalting into storage buffer.
4. The TurboLink™ Catalyst should be removed soon after the conjugation reaction is complete. The reaction
solution may either be purified by using chromatography immediately, or may be desalted into storage
buffer using either a 0.5 or 2 mL ZebaTM column based on the conjugation reaction volume (0.5 ml ZebaTM
13
column has a capacity of 50 – 150 μL; 2 ml ZebaTM column has a capacity of 150 – 700 μL).
5. If using a 0.5 mL Zeba, proceed to buffer exchange the conjugated proteins into 1X PBS Buffer as described
on page 8. For using the 2 mL ZebaTM column, please see the protocol below.
3.1 ZebaTM Column Desalting Protocol
2 mL Zeba™ Spin Column Preparation and Sample Loading (150 – 700 µL)
1. Remove spin column bottom closure and loosen the top cap (do not remove cap).
2. Place spin column in a 15 mL conical collection tube.
3. Centrifuge at 1,000 x g for 2 minutes. Discard the flow through from the collection tube.
4. Place a mark on the side of the column where the compacted resin is slanted upward. Place column in the
15 mL conical collection tube with the mark facing outward and away from the center of the rotor in all
subsequent centrifugation steps.
5. Add 1 mL of 1X PBS Buffer to the top of the resin bed and centrifuge at 1,000 x g for 2 minutes. Discard the
flow through from the collection tube.
6. Repeat step 5 an additional two times, discarding the flow through from the collection tube.
7. Place the ZebaTM column in a new 15 mL conical collection tube, remove cap, and slowly apply sample onto
the center of the compacted resin bed (150–700 µL).
Note- For sample volumes less than 350 µL, apply a 40 µL 1X PBS Buffer stacker to top of the resin bed
after the sample has fully absorbed to ensure maximal protein recovery. Avoid contact with the sides of
the column when loading.
8. Centrifuge column at 1,000 x g for 2 minutes to collect desalted sample.
9. Determine final protein concentration using BCA or Bradford assay. Add bacteriostat (e.g. 0.05% sodium
azide) and/or a protein stabilizer (e.g. 0.5% BSA) if necessary, then store at 4oC.
3.2 Analysis of the Conjugated Proteins
After completion of the conjugation reaction, a small aliquot of the crude reaction mixture is often analyzed using
a 4-12% Bis-Tris gel in an SDS (non-reducing) running buffer system. The amount of crude sample loaded on these
gels depends on the type and sensitivity of staining method being used. For example, Coomassie blue stain can
easily detect 2-4 micrograms of crude conjugate per lane. An appropriate protein marker is loaded side by side,
as well as both HyNic- and 4FB-modified proteins as references. The protein conjugate will migrate more slowly in
the gel and is visualized as higher molecular weight species (see appendix for an example).
4.0 Purification
All conjugation reactions will be a mixture of reaction products consisting of the desired conjugate along with
some un-conjugated HyNic- and 4FB-modified proteins. For this reason, some conjugation reactions are purified
using chromatographic methods. Various FPLC or HPLC chromatography workstations are available for this
purpose. Solulink routinely purifies conjugates using size exclusion or ion exchange chromatography media.
Other media such as metal chelate affinity chromatography or protein A/G columns may be used where
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appropriate.
5.0 Troubleshooting
Problem
Poor modification of protein
Possible Cause
Recommended Action
Initial protein concentration is too
low
Concentrate protein using a
diafiltration filter to 1-5 mg/mL for
efficient modification of protein
Add more modification reagent. Up
to 40 equivalents can sometimes be
added
Exchange the protein buffer by
desalting, dialysis or diafiltration
before modification
Verify using the NCBI protein
database
Make sure all solutions being used
are clear
Insufficient equivalents of
modification reagent added
Molar substitution assay readings
are out of range
Precipitation of modified protein
Protein precipitates during
conjugation reaction
Amine contaminant, e.g. Tris or
glycine buffer present in starting
biomolecule solution
The protein being modified has
insufficient amino groups
Precipitation of the modified
protein on treatment with
quantification reagents can lead to
spurious reading
Over-modification
Conjugation reaction pH may be
close to the isoelectric point of the
conjugate being formed
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Decrease equivalents of
modification reagent
Conjugate at a different pH, higher
or lower than PI (but below pH 6.5)
6.0 Appendix
To determine the best conjugation condition, we recommend that the customer perform a titration of HyNicprotein-1 with 4FB-protein-2 of different equivalents before scaling up, as show below:
Conjugation cross titration guide
HyNic-protein-1
4FB-protein-2
5X HyNic
5X 4FB
10X HyNic
10X 4FB
20X HyNic
15X 4FB
Bovine IgG
(3.0mg/ml)
MSR
BSA
(2.5mg/ml)
MSR
5X HyNic
3.2
5X 4FB
3.5
10X HyNic
4.8
20X HyNic
8.0
10X 4FB
15X 4FB
5.4
6.9
Table 3: MSR of Modified Bovine IgG and BSA
Protein-protein conjugation:
Bovine IgG was modified with 5, 10 or 20 mole equivalents of S-HyNic. BSA was modified with 5, 10 or 15 mole
equivalents of S-4FB (Table 3). Then HyNic-bovine IgG and 4FB-BSA were conjugated by each cross titration
illustrated above at ratio 1:1.5 of bIgG : BSA. After final desalting, 4ug each of crude conjugated was loaded on a
12% Bis-Tris SDS-PAGE gel, followed by Coomassie staining:
Lane 1. Protein Marker
2. HyNic-Bovine IgG (bIgG)
3. 4FB-BSA
8. 10xHyNic bIgG/10x4FB BSA
4. 5xHyNic bIgG/5x4FB BSA
9. 10xHyNic bIgG/15x4FB BSA
5. 5xHyNic bIgG/10x4FB BSA
10. 20xHyNic bIgG/5x4FB BSA
6. 5xHyNic bIgG/15x4FB BSA
11. 20xHyNic bIgG/10x4FB BSA
7. 10xHyNic bIgG/5x4FB BSA
12. 20xHyNic bIgG/15x4FB BSA
Result:
Lanes 4-6 Most of 1:1 bIgG/BSA conjugated at low mole equivalents of Hynic/4FB modified proteins.
Lanes 7-12 1:2, 1:3 and polymer of bIgG/BSA conjugated are gradually formed based on increasing
mole equivalents of Hynic/4FB modified proteins.
Note – This example conjugation is meant as an illustration using model proteins-your sample may require
modified condition to yield the desired conjugate. Please contact Solulink if you need more reagents or
assistance in choosing condition for modification/conjugation of your particular proteins.
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